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| United States Patent |
5,185,323
|
|
Gewirtz
|
February 9, 1993
|
Suppression of megakaryocytopoiesis employing platelet factor 4
antimaturation factor
Abstract
Antimaturation factor (platelet factor 4 or active peptide segments
thereof) is employed in the clinical treatment of coagulation disorders as
an anticoagulant operating via an autoregulator mechanism for selectively
suppressing megakaryocytopoiesis. Exposure of immature megakaryocytes to
antimaturation factor reversibly inhibits cell maturation and,
accordingly, functions characteristic of the mature cell, including
platelet production and expression of genes coding for platelet
coagulation factors, are reversibly suppressed.
| Inventors:
|
Gewirtz; Alan M. (Philadelphia, PA)
|
| Assignee:
|
Temple University (Philadelphia, PA)
|
| Appl. No.:
|
175552 |
| Filed:
|
March 31, 1988 |
| Current U.S. Class: |
514/12; 424/85.1; 514/21 |
| Intern'l Class: |
C07K 015/06 |
| Field of Search: |
514/12,21
530/380
424/85.1
|
References Cited
U.S. Patent Documents
| 4702908 | Oct., 1987 | Thorbecke et al. | 424/88.
|
Other References
A. Gewirtz, W. Y. Xu, B. Rueinski, and S. Niewiarowski, Highly Purified
Platelet Factor 4 Selectively Inhibits In Vitro Human
Megacaryorytopoiesis, Clinical Research, vol. 35, No. 3. Apr. 1987.
A. Gewirtz, B. Callabretta, B. Rucinski, and S. Niewiarowski, Studies on
the Mechanism of Platelet Factor 4 Induced Inhibition of Human
Megacaryocytopoiesis in Vitro; Blood, vol. 70 (Supplement #1), p. 153a,
Nov. 1990.
Gewirtz et al. Jul. 6-10, 1987, Thromb. Haemostasis 58(1):493, Abstract No.
1822.
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Furman; Keith C.
Attorney, Agent or Firm: Ratner & Prestia
Claims
I claim:
1. A method for reducing the number of circulating platelets in the
bloodstream of a mammal, comprising administering to the mammal a
sufficient amount of Antimaturation factor to effect said reduction.
2. The method of claim 1, wherein the Antimaturation factor is intact
platelet factor 4.
3. The method of claim 1, wherein the Antimaturation factor is an active
peptide segment of platelet factor 4.
4. The method of claim 3, wherein the active peptide segment is a
carboxy-terminal segment.
5. The method of claim 4, wherein the active peptide segment contains at
least about 24 amino acid residues.
6. The method of claim 1, wherein an amount of Antimaturation factor is
administered sufficient to reduce the number of circulating platelets by
at least about 10%.
7. A method for stimulating in a mammal the production of platelets
deficient in at least one platelet coagulation factor, comprising
administering to the mammal a sufficient amount of PF4 Antimaturation
factor to effect said deficiency.
8. The method of claim 7, wherein the Antimaturation factor is platelet
factor 4.
9. The method of claim 7, wherein the Antimaturation factor is an active
peptide segment of platelet factor 4.
10. The method of claim 9, wherein the active peptide segment is a
carboxy-terminal segment.
11. The method of claim 10, wherein the active peptide segment contains at
least about 24 amino acid residues.
12. The method of claim 7, wherein the coagulation factor is cofactor V.
13. The method of claim 12, wherein the Antimaturation factor is platelet
factor 4.
14. The method of claim 12, wherein the Antimaturation factor is an active
peptide segment of platelet factor 4.
15. The method of claim 1, wherein at least some of the circulating
platelets after said administration are deficient in at least one platelet
coagulation factor.
16. The method of claim 15, wherein the deficient factor is platelet
coagulation cofactor V.
17. The method of claim 16, wherein the Antimaturation factor is platelet
factor 4.
18. A method for the clinical treatment of a thromboembolic disease or
disorder, comprising administering to a human affected with said disease
or disorder an anticoagulant comprising Antimaturation factor in an amount
sufficient to reduce the thromboembolic component of said disease or
disorder.
19. The method of claim 18, wherein the Antimaturation factor is
administered in an amount sufficient to reduce the number of platelets
circulating in the bloodstream of said human, or to reduce the coagulant
potential of said platelets, or both.
20. The method of claim 19, wherein the Antimaturation factor is intact
platelet factor 4.
21. The method of claim 19, wherein the Antimaturation factor is an active
peptide segment of platelet factor 4.
22. The method of claim 21, wherein the active peptide segment is a
carboxy-terminal peptide segment.
23. The method of claim 22, wherein the active peptide segment contains at
least about 24 amino acid residues.
24. A method for suppressing megakaryocytopoiesis in a mammal, comprising
administering to the mammal a sufficient amount of Antimaturation factor
to inhibit at least one of maturation of immature magakaryocyte progenitor
cells and maturation of immature megakaryocyte precursor cells.
25. The method of claim 24, wherein the Antimaturation factor is intact
platelet factor 4.
26. The method of claim 24, wherein the Antimaturation factor is an active
corresponding peptide segment of platelet factor 4.
27. The method of claim 24, wherein the Antimaturation factor is an active
homologous peptide segment of intact platelet factor 4.
Description
BACKGROUND OF THE INVENTION
Pluripotent hematopoietic stem cells are activated in the bone marrow to
proliferate and differentiate into mature megakaryocytes, each of which is
capable of releasing up to several thousand functional platelets in
response to biological demand. Development of the stem cell proceeds by
stages broadly corresponding to proliferation of progenitor cells, and
differentiation of late progenitor and early precursor cells into mature
megakaryocytes. Although regulation of this developmental process
(megakaryocytopoiesis) is of substantial clinical interest for its
potential application to disorders characterized by abnormal platelet
production, endogenous factors responsible for stimulating or inhibiting
proliferation and differentiation of megakaryocyte progenitor/precursor
cells have not been thoroughly elaborated.
In particular, inhibition factors capable of clinically significant
megakaryocyte suppression have not been well-characterized. For example,
both immunocytes and transforming growth factor-.beta.(TGF-.beta.) have
been studied as potential inhibitors of megakaryocytopoiesis, with
inconclusive results (see, e.g., Blood 67: 479-483 and 68: 619-626, 1986).
Additionally, autoregulation via negative feedback mechanisms involving
megakaryocyte products, including plateletsecreted 12-17kD glycoprotein,
has been reported (J. Cell Physiol. 130: 361-368, 1987). While the
potential utility of negative autocrine regulators or other
megakaryocytopoiesis inhibitors in the clinical treatment of disorders
characterized by excessively high platelet counts is apparent, none of the
heretofore postulated inhibitors has so far proved useful in such
applications.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 graphically illustrates the effect of highly purified platelet
factor 4 (PF4) on clonogenicity of normal human megakaryocyte progenitor
cells in plasma clot culture. Bone marrow mononuclear cells (MNC) were
isolated by density gradient sedimentation and then suspended in the
appropriate culture medium, to which was added PF4 in the amounts
indicated. The cell suspensions were plated, and cultured for twelve days
at 37.degree. C. in a humidified environment containing 5% CO.sub.2, and
resulting megakaryocyte colonies were then enumerated in situ with an
indirect immunofluorescence assay using a polyspecific antiplatelet
glycoprotein antiserum as probe. Aggregate results (mean.+-.S.D.) of five
separate experiments are shown. Numbers at top of bars indicate p value in
comparison to control (CONT.).
FIG. 2 is a composite photomicrograph of normal human megakaryocytes probed
for expression of .beta.-actin and coagulation cofactor V (FV) mRNA by in
situ hybridization with DNA probes labeled by random priming with
biotin-11-dUTP, illustrating the effect of PF4 on expression of FV mRNA in
human megakaryocytes. Megakaryocytes were separated from normal human bone
marrow by counterflow centrifugal elutriation, and then placed in
suspension cultures for sixteen to eighteen hours in medium containing
either a synthetic "long" PF4 peptide (100 .mu.g/ml) or no PF4. DNA-RNA
hybridizations are indicated by the appearance of purple-brown precipitate
over the cytoplasm of the cell; the greater the amount of hybridization
that occurs, the darker and denser the precipitate that forms. Appearance
of cells post-hybridization with probes for pBR322 and 8-actin is shown in
Panels A and B, respectively. Panel C demonstrates the appearance of cells
pre-treated with RNase A (500 .mu.g/ml) for .about.on hour prior to
hydridization with 8-actin probe. Panels D and E illustrate representative
color development for megakaryocytes hybridized with FV probe (note
(arrows) two small, unlabeled mononuclear cells on Panel D megakaryocyte).
Panel F shows representative color occurring in megakaryocytes hybridized
with FV probe after sixteen to eighteen hour incubation in PF4.
FIG. 3 graphically illustrates the effect of highly purified platelet
factor 4 (PF4) on normal human megakaryocyte colony formation in vitro in
the absence of accessory marrow immunocytes. Isolated bone marrow
mononuclear cells (MNC) were depleted of adherent monocyte-macrophages and
lymphocytes and treated as detailed in the Examples. Each line represents
the result of a single experiment performed in quadruplicate culture
plates. p values in comparison to control are indicated for each
experiment.
FIG. 4 graphically illustrates the effect of highly purified platelet
factor 4 (PF4) on clonogenicity of normal human erythroid colony forming
units (CFU-E) in plasma clot culture in the absence of accessory marrow
immunocytes. Isolated bone marrow mononuclear cells (MNC) were depleted of
adherent monocyte-macrophages and lymphocytes, as detailed in the
Examples. After seven days in culture, plates were harvested and fixed and
CFU-E detected by benzidine staining. Results of three separate
experiments, each performed in quadruplicate culture plates, are shown.
BRIEF DESCRIPTION OF THE INVENTION
According to the invention, platelet factor 4 (PF4), a
megakaryocyte/platelet-specific .alpha.-granule protein, has been
identified as a negative autocrine regulator with clinical utility in the
treatment of coagulation disorders characterized by excessively high
platelet counts. Both PF4 and active PF4 peptide segments suppress
megakaryocyte maturation (differentiation) without significant inhibitory
effect on progenitor/precursor cell proliferation, and are characterized
herein as selective megakaryocyte Antinaturation factors which inhibit
development of immature late progenitor/early precursor cells into
platelet-producing mature megakaryocytes.
It has additionally been found that PF4 and active peptide segments thereof
(herein generically referred to as Antimaturation factor) inhibit the
expression of genes coding for platelet coagulation factors in
megakaryocytes. Exposure of immature megakaryocytes to Antimaturation
factor thus results in mature cells which produce platelets deficient in
these coagulation factors and which cannot, therefore, fully participate
in the normal coagulation "cascade."
Antimaturation factor accordingly comprises an anticoagulant potentially
useful in the treatment of a variety of coagulation disorders associated
with platelet/platelet coagulation factor overabundance. Since PF4
Antimaturation factor is synthesized by platelets, and suppresses
megakaryocyte maturation in a reversible and saturable manner, PF4
Antimaturation factor is a bona fide negative autoregulator of
megakaryocytopoiesis and, accordingly, safe, effective clinical use is
contemplated.
DETAILED DESCRIPTION OF THE INVENTION
According to the invention, PF4 and active peptide segments thereof are
employed to inhibit megakaryocytopoiesis to effect reduction of platelet
production in vivo, as measured by blood platelet count, and to inhibit
production of platelet coagulation factors. Sufficient PF4 or active
peptide segment is employed to effect the desired reduction in platelet
count, preferably by intravenous administration. Depending upon the route
of administration and idiosyncratic factors, particularly individual
platelet counts and rates of Antimaturation factor clearance, average
dosages of up to about 12 gms active material per day in humans are
contemplated.
Both PF4 and active peptide segments thereof are useful in the process of
the invention as PF4 anti-maturation factor. PF4 is a known
.alpha.-granule protein specific to platelets, which exists in the
unactivated platelet as a granulated tetramer composed of identical 7800
kD monomers, as more fully described in The Platelet's Physiology and
Pharmacology, Niewiarowski, et al. (Academic Press Inc. N.Y. 1985),
especially pp. 49-83 thereof; Proc. Nat'l Acad. Sci. USA 74: 2256-2258
(1977); J. Biol. Chem. 252: 6276-6279 (1977); and Biochim. Biophys. Acta
286: 312-329 (1972) PF4 is readily obtained by purification from human
platelets according to published procedures (see, e.g., Blood 53: 47-62,
1979). As used herein, the term Antimaturation factor includes active PF4
peptide segments having amino acid sequences corresponding or homologous
to amino acid sequences of the PF4 protein molecule, referred to as "PF4
corresponding peptide segments" and "PF4 homologous peptide segments,"
respectively. The term "active PF4 peptide segments" accordingly
generically includes PF4 corresponding peptide segments and PF4 homologous
peptide segments, wherein one or more amino acids in a PF4 corresponding
peptide segment are substituted with a different amino acid, with the
proviso that the PF4 peptide segment functions to suppress
megakaryocytopoiesis and/or inhibit megakaryocyte maturation; i.e., is
"active" within the scope of the invention. The segments may be derived
from naturally-occurring proteins, or be synthesized according to
well-established procedures. Since it is believed that the inhibitory
activity of PF4 peptide segments may be localizable to the
carboxy-terminal domains thereof, such c-terminal segments are presently
preferred. Generally, greater activity tends to be found in segments
containing at least about 24 carboxy-terminal amino acid residues (e.g.,
at least residues #46-70 of PF4), and the use of such segments is
accordingly recommended.
Antimaturation factor according to the invention is contemplated for use in
lowering blood levels of circulating platelets as deemed clinically
advantageous, and for use in reducing the ability of these platelets to
support blood clot formation. As described in the Examples herein, in
addition to suppressing megakaryocyte platelet production, a function of
the mature cell, PF4 Antimaturation factor additionally suppresses
megakaryocyte expression of mRNA coding for platelet procoagulation
factors, especially platelet coagulation cofactor V (FV). Since production
of cofactor V is also a characteristic of mature megakaryocytes, platelet
deficiency in this cofactor, after exposure of the parent megakaryocyte to
PF4 Antimaturation factor, both evidences the antidifferentiation function
of PF4 and reinforces the anticoagulant activity thereof.
Pathological vascular reactions associated with excessively high platelet
counts include stroke, pulmonary emboli, and related thromboembolic
complications. A predisposing factor for these potentially fatal
complications, high circulating platelet levels, can be substantially
minimized by administration of Antimaturation factor according to the
invention. Since PF4 Antimaturation factor also inhibits production of
coagulation cofactor V, the therapeutic use of this PF4 factor also
stimulates the production of platelets less able to support blood clot
formation, and is therefore broadly indicated in the treatment of diseases
or disorders with a prominent thromboembolic risk, whether or not
accompanied by high platelet counts. The invention is of particular
clinical relevance in the treatment of myeloproliferative and other
disorders characterized by clinically disadvantageous high platelet
counts, as well as coagulation disorders more broadly characterized by a
high thromboembolic component with attendant risk of heart attack, venous
thrombosis, stroke, pulmonary emboli, or related vascular accidents.
Treatment of such diseases or disorders is accomplished according to the
process of the invention by the administration of PF4 or active peptide
segments thereof in sufficient quantities to suppress platelet production
and approach normal hemostasis, as measured by significant reduction in
blood platelet count. Generally, a clinically significant effect comprises
a reduction in platelet count of at least about 10%. Contemplated routes
of administration include intravenous and parenteral routes, including per
os. Therapeutic dosages of up to about 12 grams Antimaturation factor per
day per 150 lbs. body weight comprise a general guideline for effecting
the desired platelet count reduction or change in procoagulant activity.
In view of the previously described immunomodulatory activity of PF4 (see,
e.g., J. Immunol. 134; 3199-3203, 1985), the efficacy of the process of
the present invention is unexpected. In particular, it has been reported
that platelet .alpha.-granule releasates augment immune response in mice
(J. Immunol., ibid.) and further, that CD4.sup.+ "helper" T lymphocytes
promote megakaryocytopoiesis (see, e.g., J. Immunol. 137: 2508-2513, 1986;
Blood 68: 991-995, 1986). Accordingly, PF4 augmentation of
megakaryocytopoiesis would be expected. To the contrary, as described
herein, PF4 suppresses megakaryocytopoiesis. Moreover, in contrast to
prior art inhibitors such as TGF-.beta., which suppress hematopoiesis in a
more general response, Antimaturation factor specifically inhibits
megakaryocytopoiesis in a reversible, saturable manner. As a selective
endogenous auto-regulator of megakaryocytopoiesis, PF4 Antimaturation
factor not utilized in megakaryocyte suppression is readily cleared from
the blood. Further, the reversibility of the reaction ensures that dosages
can be optimized on a trial-and-error basis without risk of irreversible
side-effects, and the selectivity of the factor minimizes potential
involvement of related pathways. Accordingly, PF4 Antimaturation factor is
contemplated for use as a safe, efficacious therapeutic for suppression of
platelet production and platelet coagulation factor determinants by
megakaryocyte target cells.
EXAMPLES
Methods and Materials
A. Preparation of Antimaturation Factor
Purification of PF4 and Synthesis of Synthetic C-terminal PF4 Active
Peptide Segments
Human PF4 was ified essentially as published (Blood 53: 47-62, 1979). In
brief, outdated human platelets were thrombin-stimulated. The resulting
supernatant was applied to a heparin-agarose column. The column was
washed, and the PF4 specifically eluted with 1.5M NaC1. Some PF4
preparations were purified from the supernatants of stored, outdated
platelets by a combination of Sephadex G-75 gel filtration, and heparin
a9arose column chromato9raphy. Eluted fractions were pooled, and then
stored lyophilized. Such preparations gave a sin91e band on
SDS-Polyacrylamide gels, and were judged to>95% pure.
Synthetic PF4 peptides were commercially synthesized and purified
(Pennisula Laboratories, Belmont, CA). Purity and sequence were confirmed
by the Macromolecular Analysis and Synthesis Laboratory, Temple University
School of Medicine, by Edman Degradation using Applied Biosciences
instrumentation. The peptides utilized consisted of either the terminal 13
(amino acid residues #58-70), or terminal 24 amino acids (residues
#46-70) in the 70 amino acid human PF4 sequence. These were designated
"short" and "long" PF4 peptide, respectively.
B. Cells and Tissue Culture Methods
Megakaryocyte Cell Assay
Megakaryocyte colonies were cloned in plasma clot cultures as previously
described (e.g., Blood 61: 384-9, 1983). The cell population cultured
consisted of either unseparated high density marrow mononuclear cells
(MNC), or MNC depleted of adherent monocytemacrophages (MO) and T
lymphocytes using methods previously reported (e.g., J. Immunol.
2915-2925, 1987). To provide essential growth factors, all cultures were
supplemented with normal human AB serum (30% v/v) derived from the
platelet-poor plasma of a single donor.
Megakaryocyte colonies were enumerated by indirect immunofluorescence assay
utilizing a rabbit anti-human platelet glycoprotein antiserum as a
megakaryocyte probe (ibid.). The antiserum utilized is highly specific for
recognition of platelet glycoproteins, and it does not recognize
monocytes. A cluster of three or more intensely fluorescent cells was
counted as one colony. Unless otherwise stated, all data are reported as
the mean.+-. S.D. of colonies enumerated.
Isolation of Mature Marrow Megakaryocytes
Mature (morphologically recognizable) megakaryocytes were isolated from the
marrow of normal bone marrow donors by the process of counterflow
centrifugal elutriation (CCE), as previously described (e.g., Blood 37:
1639-1648, 1986). Such cells were then utilized for culture, or suspended
in Supplemented Alpha medium containing 5% normal human AB serum (derived
from the platelet poor plasma of a single donor) and subjected to
short-term culture, either in the presence or absence of 100 .mu.g/ml of
synthetic "long" C-terminal PF4 peptide (see below). After culture, cells
were fixed in 4% paraformaldehyde, and stored in 70% ethanol at 4.degree.
C. for use in situ hybridizations (see below).
C. Molecular Analysis Methods
Isolation of RNA
Total cellular RNA was purified from cells as previously described (ibid.)
In brief, cells were homogenized in a Waring blender in nucleic acid
extraction buffer (75mM NaCl, 20mM EDTA, 10mM Tris-HCL, ph 8.0, and 0.2%
sodium dodecyl sulfate), mixed 1:1 with buffer-saturated phenol. The
aqueous phase was recovered by centrifugation, re-extracted with an equal
volume of phenol and chloroform:isoamyl alcohol (25:24:1), and finally
with chloroform:isoamyl alcohol (24:1). Nucleic acids were precipitated
with two-and-one-half volumes of ethanol, and DNA removed by treatment
with DNase 1 and precipitation with 3M sodium acetate (pH 5.5). The
integrity and amount of RNA samples were monitored by ethidium bromide
staining of agarose-formaldehyde gels.
In situ Hybridization Procedure
In situ hybridization was performed using a synthesis of the techniques
described in Virology 126: 32-50, 1983 and Biotechniques 4: 39-39 230-250,
1986. Human megakaryocytes, fixed and stored as described above, were
deposited onto glass slides by cytocentrifugation (500 rpm.times.8
minutes, Cytocentrifuge II, Shandon Southern, Sewicky, PA).
Prehybridization washes, including treatment with acetic anhydride (0.1%
in triethanolamine), were carried out as described (ibid.). Hybridization
was carried out using DNA probes oligolabeled with biotin-11-dUTP (BRL,
Gaithersburg, MD) using the method of Ann. Biochem. 137: 266-267, op. cit.
25 .mu.l of probe [(100ng/ml of hybridization cocktail containing 45%
formamide (Amersco, Solon, Ohio)] was layered over the specimen which was
then covered with a glass slide, sealed in parafilm, and then hybridized
for .about.18 hours at 37.degree. C. Post-hybridization washes were
carried out as described in Biotechniques 4: 32-39, op. cit., the final
wash being carried out in 0.16% SSC at room temperature. DNA-RNA hybrids
were detected with a DNA Detection Kit, essentially as described by the
manufacturer (BRL, Gaithersburg, MD). Positive reactions consisted of a
purple to deep brown colored precipitate in the cell's cytoplasm. The
degree of hybridization correlates directly with the amount of precipitate
accumulated in the cell.
Hybridization with pBR322 was carried out as a negative control. An
additional control consisted of pre-treating specimens with RNase A (500
.mu.g/ml) prior to hybridization with the probe of interest. Hybridization
with a cDNA coding for human .beta.-Actin was employed as a positive
control.
Reactions were semi-quantitated by computer assisted microspectrophotometry
(Zonax, Carl Zeiss, Mineola, N.Y.) as a function of light transmission
through the object cell. The photometer was standardized so that light
transmission through a clear area of the slide containing no cells
(background) was defined as 100% transmission, while no light falling on
the photometer was defined as 0% transmission. Identical gain and high
voltage setting were employed throughout.
D. Plasmids
Plasmids containing the cDNA inserts used as probes in these experiments
have been previously described pUC9 carrying the human coagulation factor
V (FV) gene probe was provided by University of Washington, Seattle,
Washington. pBR322 was obtained from the American Type Culture Collection
(Rockville, MD). The Department of Pathology, Temple University Medical
School, supplied plasmids containing human .beta.-actin inserts.
E. Statistical Analysis
Statistical significance of differences between groups was tested using a
two-tailed Student's T Test for unpaired observations.
EXAMPLE I. Characterization of PF4 and active PF4 peptide segments as
Antimaturation factor
I.A. Effecto of PF4 on megakaryocyte colony formation (proliferation
assay).
To determine if PF4 could modify megakaryocyte colony formation in vitro, a
screen assay was performed by adding various amounts of pure protein to
unseparated marrow MNC. To emulate basal growth conditions in marrow, the
cultures contained no exogenous source of growth factors, and were
supplemented only with normal human AB serum derived from platelet-poor
plasma The aggregate results of five such experiments are shown in FIG. 1.
In the control condition, .about.11 colonies per 2.times.105 MNC plated
were enumerated. In the presence of 2.5 .mu.g/ml of PF4, .about.7 colonies
were counted. This difference was not significant (p>0.05). However, in
the presence of 25 .mu.g/ml of PF4, a mean of five colonies was enumerated
and this difference was highly significant (p<0.007).
I.B Effect of synthetic c-terminal PF4 peptides on megakaryocyte colony
formation (proliferation assay).
These preparations were employed to exclude from consideration that the
inhibitory effect observed in Example IA might be due to impurities in the
PF4 preparation, and to determine whether the colony inhibitory effect
observed might be localizable to the c-terminal domain of PF4. Also, since
secondary and tertiary structure are important in mediating a protein's
biological activities, it was of interest to obtain structure/function
information by employing these peptides in the described bioassay system.
One peptide consisted of the last 13 amino acid residues from the carboxy
terminus and was designated "short peptide." The other consisted of the
last 24 residues and was designated "long" peptide.
The results of four experiments with the synthetic c-terminal peptides are
shown in Table 1. As indicated, the short and long peptides were tested in
a dose response manner at 2.5, 25 and 50 .mu.g/ml concentrations. The
short peptide demonstrated inhibitory activity in only one of the three
studies performed, and this was at the highest concentration tested. In
contrast, inhibitory activity was more consistently noted with the long
peptide.
TABLE 1
__________________________________________________________________________
Effect of Synthetic C-terminal PF4 Peptides on
Megakaryocyte Colony Formation
Light density marrow mononuclear cells were depleted of adherent
monocyte-
macrophages and T lymphocytes, and then cloned in plasma clot cultures
as
described in the Experimental Procedures section. "Short" (13 AA
residues)
and "Long" (24 AA residues) synthetic PF4 peptides were then added to
the
cultures in dose response fashion and resulting megakaryocyte colonies
enumerated as described.
SHORT PEPTIDE LONG PEPTIDE
STUDY #
Control (C) Cells
2.5 .mu.g/ml
25 .mu.g/ml
50 .mu.g/ml
2.5 .mu.g/ml
25 .mu.g/ml
50 .mu.g/ml
__________________________________________________________________________
1 .sup. 31 .+-. 1.sup.
31 .+-. 5
33 .+-. 2
28 .+-. 4
37 .+-. 5
23 .+-. 2
9 .+-. 6
(p = .05).sup.+
(p = .05)
2 113 .+-. 12
157 .+-. 12
107 .+-. 10
94 .+-. 8
88 .+-. 14
144 .+-. 17
50 .+-. 26
(p = .05)
3 7 .+-. 1
5 .+-. 0
NT* 1 .+-. 0
8 .+-. 2
NT 1 .+-. 1
(p = .01) (p = .01)
4 24 .+-. 5
NT NT NT 18 .+-. 4
10 .+-. 2
10 .+-. 1
(p = .03)
(p = .02)
__________________________________________________________________________
.sup. Mean .+-. SEM of megakaryocyte colonies enumerated in quadruplicate
culture plates
.sup.+ p statistic in comparison to growth in control plates
*NT = not tested In two of three experiments, long peptide inhibited
colony formation at 25 .mu.g/ml concentration, and in four experiments at
the 50 .mu.g/ml concentration. These results indicate that results with
the purified PF4 protein were not due to artifact and that the
carboxyterminal domain of the protein contains at least part of the
inhibitory activity noted in the above experiments.
I.C. Effect of PF4 carboxy-terminal segment on megakaryocyte maturation and
progenitor cell proliferation.
First, to assess potential effects on progenitor cell proliferation, the
total numbers of cells comprising each individual colony in two hundred
control colonies and in one hundred colonies cloned in plates containing
"long" PF4 peptide were enumerated. Control colonies were found to contain
6.1.+-.3.0 (mean .+-.S.D) cells per colony, while colonies grown in the
PF4 containing plates contained 4.2+1.6 cells per colony. This difference
was small, but of statistical significance (p<0.001) (Table 2). The number
of "large" (mature) cells and "small" (immature) cells in these same
colonies were then quantitated as an index of effect on cell maturation
(Table 2). Control colonies were composed of 3.9.+-.2.3 large cells, while
those arising in PF4 containing plates had 1.6.+-.1.6 large cells. This
59% reduction in large cells was highly significant (p<0.001). In
contrast, there were 2.1.+-.2.1 small cells in control colonies in
comparison to 2.6.+-.1.8 in plates containing PF4. In aggregate, these
results demonstrate that PF4 exerts a greater effect on megakaryocyte
maturation than on megakaryocyte progenitor cell proliferation.
TABLE 2
______________________________________
Effect of Long Synthetic C-terminal PF4 Peptide on
Megakaryocyte Maturation and Progenitor Cell
Proliferation
Marrow mononuclear cells were prepared and cultured as
described in the legends for Tables 1 and 3 and in
"Methods and Materials." Long PF4 was added at a
final concentration of 50 .mu.g/ml. Colonies were identi-
fied by indirect immunofluorescence and analyzed in
situ at total magnifications of 100X and 400X.
Colonies Cloned
Control Colonies
in PF4
(n = 200).sup.
(n = 100)
______________________________________
Total Cells/Colony
.sup. 6.1 .+-. 3.0.sup.+
4.2 .+-. 1.6*
"Large" Cells/Colony
3.9 .+-. 2.3 1.6 .+-. 1.6*
"Small" Cells/Colony
2.1 .+-. 2.1 2.6 .+-. 1.8
______________________________________
.sup. n = total number of colonies examined
.sup.+ Mean .+-. SD of total cells enumerated
*p < .0001
I.D. Effect of PF4 and a carboxy-terminal active peptide segment thereof on
megakaryocyte maturation employing FV as marker (maturation assay).
Coagulation cofactor V (FV) was chosen as a suitable marker since this
protein is expressed only in more mature cells of the megakaryocyte
lineage. Normal, mature human megakaryocytes were isolated from bone
marrow by centrifugal elutriation, and suspended for up to twenty-four
hours in liquid cultures containing 100 .mu.g/ml of synthetic "long" PF4
peptide. The cells were then fixed as described above and probed for the
expression of FV mRNA by the technique of in situ hybridization using a
biotinylated cDNA probe. Results of typical experiment are shown in a
composite photomicrograph (FIG. 2, Panels A-F). Cells in Panel A were
hybridized with a pBR322 probe and are unlabeled. In Panel B, cells were
probed with an insert for human .beta.-actin and are strongly labeled.
This signal is essentially eliminated by pre-treating cells with RNase
(500 .mu.g/ml) prior to hybridization as shown in Panel C Panels D-E show
typical signal achieved after hybridization with the FV cDNA probe. Panel
F demonstrates the marked reduction in signal noted after incubation of
PF4 for 16-18 hours. Dose response testing revealed comparable decreases
in FV mRNA expression at concentrations of long peptide to 100 ng/ml. At
20 ng/ml, the effect was no longer detectable.
Accumulation of indicator dye was semiquantititated by
microspectrophotometry as described under "Materials and Methods." Since
increasing amount of hybridization correlates directly with the density of
dye accumulation in a given cell, and increasing dye accumulation impedes
light transmission through any given cell being examined, those cells
which allow the greatest degree of light transmission are expressing the
least amount of message. It is emphasized that, since the photometer is
not as sensitive to grey scale changes within a color family as it is to
black and white, differences recorded are underestimates of true changes
observed (FIG. 2).
Light transmission through mononuclear cells was found to be quite constant
after hybridization with the FV probe, regardless of the conditions under
which the cells were cultured. MNC in the control suspension cultures
(number examined [n]=228) were found to allow 73.2.+-.7.5% light
transmission (100% maximum), while similar cells exposed to 100 .mu.g/ml
of synthetic PF4 peptide permitted 72.8+7.8% light transmission ([n]=216).
These differences were of no statistical significance (p=0.559). In
contrast, light transmission through megakaryocytes incubated in PF4 was
64.4.+-.8.4% post-hybridization ([n]=292) with the FV cDNA probe, versus
58.+-.9.5% in the control cells ([n]=298). This difference, while small in
absolute terms, was highly significant (p<.001). If the change in light
transmission in these groups is compared to that permitted by the
"unlabeled" MNC, it is calculated that control megakaryocytes had 60%
greater dye accumulation than cells incubated in PF4 [73%-68%=.DELTA.9;
73%-58%=.DELTA.15; 9/15=.6]. Since these differences are underestimates of
true change in color, these data are indicative of a highly significant
decline in FV mRNA, post-exposure to PF4.
EXAMPLE II. Characterization of Antimaturation factor activity as non-T
lymphocyte dependent: effect of T cells and T cells plus PF4 on
megakaryocyte colony formation.
Since the majority of marrow T lymphocytes are of the suppressor type, and
PF4 is described in the prior art as inactivating these cells, the results
obtained in Examples IA and IB were unexpected. In fact, it was
anticipated that colony formation might be increased because of unopposed
T helper activity. Putative progenitor cell-T cell-PF4 interactions were
accordingly studied. Control target cells, depleted of adherent monocytes
and T lymphocytes were cloned in plasma clots under standard conditions
(Table 3). Aliquots of these target cells were also cloned in the presence
of either CD4.sup.+ helper, or CD8.sup.+ suppressor T cells alone, or T
effector cells plus PF4 at two different concentrations, 2.5 and 25
.mu.g/ml.
TABLE 3
__________________________________________________________________________
Effect of Co-Culturing Human Megakaryocyte Progenitor Cells and T
Lymphocyte
Subsets in the Presence of Different Amounts of Highly Purified PF4
Light density marrow mononuclear cells were depleted of adherent
monocyte-
macrophages, and T lymphocytes. They were then cloned in plasma clots (2
.times. 10.sup.5 /ml)
with autologous T lymphocyte subsets, in the presence of varying amounts
of purified
PF4 as described in Experimental Procedures. Megakaryocyte colonies were
enumerated
in situ by indirect immunofluorescence.
C/T4 C/T4 C/T8 C/T8
2:1 + 2:1 + 2:1 + 2:1 +
C/T4 PF4 PF4 C/T8 PF4 PF4
STUDY #
Control (C) Cells.sup.
2:1.sctn.
2.5 .mu.g/ml
25 .mu.g/ml
2:1 2.5 .mu.g/ml
25 .mu.g/ml
__________________________________________________________________________
1 .sup. 7 .+-. 2.sup.+
11 .+-. 0
8 .+-. 1
NT.sup.#
6 .+-. 1
18 .+-. 2
NT
2 4 .+-. 1 3 .+-. 1
5 .+-. 1
NT NT NT NT
3 11 .+-. 3 10 .+-. 0
NT NT NT 7 .+-. 2
1 .+-. 0*
4 116 .+-. 6
141 .+-. 22
122 .+-. 29
8 .+-. 1*
163 .+-. 13
87 .+-. 15*
13 .+-. 2*
5 33 .+-. 6 39 .+-. 4
NT 3 .+-. 1*
NT 33 .+-. 4
3 .+-. 1*
__________________________________________________________________________
.sup. Monocyte-macrophage, and lymphocyte depleted marrow mononuclear
cells 2 .times. 10.sup.5 /ml
.sctn. Ratio of Control cells to T lymphocytes
.sup.+ Mean .+-. SEM of colonies enumerated in quadruplicate culture
plates
.sup.# NT = not tested
*p < .05
In agreement with previous results from our laboratory J. Immunol. 139:
2915-2924, 1987], adding nonactivated CD4.sup.+ (helper) cells had
inconstant effects on megakaryocyte colony formation. These results were
unchanged by the addition of PF4 at concentration of 2.5 .mu.g/ml.
However, consistent with the results obtained in EXAMPLE I (FIG. 1), the
addition of high concentration of PF4 resulted in a highly significant
decrease in colony formation.
These results show the colony suppressive effect of PF4 was indifferent to
the presence of T lymphocytes, implying that PF4 has a direct suppressive
effect on colony formation.
To determine if PF4 had a direct suppressive effect on the
growth/maturation of megakaryocyte progenitor/precursor cells, purified
material was added to culture plates containing marrow mononuclear cells
depleted of adherent monocytes and T lymphocytes. The results of four
experiments of this type are shown in FIG. 3. As indicated, at the 25
.mu.g/ml concentration colony formation was greatly inhibited in each of
the experiments. The mean inhibition was .about.78%.
EXAMPLE III. Characterization of PF4 and active carboxy-terminal segment as
megakaryocyte-specific: effect of PF4 on erythroid colony formation
(specificity assay).
The effect of purified protein on inhibition of erythroid colony formation
was tested. As shown in FIG. 4, no inhibition of erythroid colony
formation units (CFU-E) was noted at concentrations of PF4 which
consistently inhibited megakaryocyte colony formation. These results
indicate that the effect of PF4 is lineage specific.
EXAMPLE IV. Exemplary Clinical Protocol
Contemplated guidelines for the clinical use of Antimaturation factor are
as follows:
5 gm Antimaturation factor is administered intravenously or by mouth in the
form of PF4 purified as described under "Methods and Materials," supra.,
to a 150 lb. male patient having distal ischemia, stroke, or other
thromboembolic phenomena attributed to abnormal platelet count or
function. The platelet count and platelet function are monitored from
seven to ten days after administration by analysis of blood samples taken
at four-hour intervals to evaluate PF4 potency (which may vary with the
donor) and blood clearance rate. At the end of the evaluation period, the
dosage is adjusted as necessary to establish an improved platelet count or
function, and the patient is again monitored once or twice weekly, as
described. At the end of this period, PF4 dosage is again adjusted as
necessary, with repetition of the described monitoring and evaluation
procedures until the platelet count is substantially stabilized at a
normal or near-normal level or the procoagulant function of the platelet
is lessened to the desired degree. The dosage required to obtain the
desired stabilized platelet count comprises a therapeutic dosage according
to the invention.
Owing to a short half-life (measurable in minutes in rodents) of
Antimaturation factor in the bloodstream, administration of the
therapeutic dosage on a daily basis to maintain platelet levels may be
necessary.
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